The Research Of Efficacy And Mechanism To Treat Inhalation Injury By Transplating BMSCs Which Were Modified By NMⅡ Gene

BackgroundInhalation injury has caused a huge disaster in the history of human development.The fire named Cocoanut Grove nightclub fire happened in Boston in 1942, the patients inhaled a lot of smoke in the closed spaces, containing carbon particles and irritating chemical substances. About 492 people were killed,despite having been rescued,the results were caused by inhalation injury. Shanghai November 15, 2010, a major fire Jiaozhou Road, Jing’an District, Shanghai medical facilities to treat the wounded 127 people, 41 people were killed, 71 people were admitted to hospital treatment.The most important deaths result was that patients had inhalation injury, which morbidity and mortality is very high.Bone Mesenchymal stem cells（BMSC） have self-renewal and differentiation potential of pluripotent stem cells,immune reaction occurred fewer,having immunolgy regulation and anti-inflammatory activity, the study found that it can induce a variety of organizations to differentiation, such as bone, cartilage, and so on.It has a strong ability of endocrine and immune function, is an ideal source of tissue repairing.The recent study have confirmed that BMSC transplantation could improve acute lung injury（ALI） and pulmonary inflammatory response of the body by regulating ALI immune disorders, "homing" BMSC to the injury lung area, the lung function of cells and play a corresponding function, participating the repair of damaged lung tissue, which is considered that it is the promising treatment of acute lung injury.Although the treatment of ALI by BMSC transplantation has a good effect, but a lot of factors effect transplantation and survival rates in vivo, the necrosis or apoptosis is the main factor, therefore, increasing or improving BMSC transplantation transplantation and survival rate in vivo is most important to the treatment. more and more researchers have proposed many theories, exploring a variety of ways in order to improve the transplantation of BMSC transplantation and survival rate.Myosin is a globulin, a major component of molecular muscle, muscle contraction provides a force mainly having enzymatic activity, through interaction ATP hydrolysis terminal phosphate group and GTP, CTP, etc. Chemical energy transform into mechanical energy.Non-muscle myosin Ⅱ（non-muscle myosinⅡ, NMⅡ）is non-muscle protein family, reported in the references,which is the key to many diseases. NMⅡin the non-muscle tissue become molecules involved in a variety of physiological activities of cells, in signal transduction, which is a key protein in cells of normal physiological activity, having a very important role and research value.In our study, we select NMⅡ to study.We found that the reduction of bone marrow mesenchymal stem cells in muscle myosin NMⅡ, and then transplanted to smoke model of acute lung injury in our preliminary study, we found that comparing its effects wih the simple injected the mesenchymal stem cells better. Further research found obvious effecttion that the gene regulation NMⅡ could make bone marrow mesenchymal stem cell transplantation decrease apoptosis and reduce inflammation than no-regulaition NMⅡ bone marrow mesenchymal stem cells. Through the series of experiments, we found that down NMⅡ gene expression could inhibit stem cell apoptosis and promote bone marrow mesenchymal stem cells homing to the site of injury, reduce injury site inflammation, bone mesenchymal stem cells are more effective than traditional, simple to use BMSC, especially lung damage having capillaries extensive more effective.Further study in NMⅡ gene,it could become a new research targets by stem cell therapying inhalation injury and ALI/ARDS in clinical application transformation.Part I: Preparation and characterization of bone marrow mesenchymal stem cellsObjective: According to the purpose of the research, prepared and identified bone marrow mesenchymal stem cells,which provides carrier for NMⅡ study of gene therapy in the efficacy and protection mechanisms inhalation injury model.Methods: Rat bone marrow mesenchymal stem cells isolated and cultured as follows: Select four weeks old, male rats weight about 160g-200 g, isolated rat femur and tibia to obtain bone marrow cells,washing from 200 mesh filter dish.Culturing at 37°C and 5% CO₂ 2-3 generations,identified surface antigen identification,ifferentiation potential ideedacntification.Results: We could see under the microscope to adherent cell morphology of different sizes, forms and kinds, most of the growth into a spindle morphology similar to fibroblasts; flow cytometry phenotyping match bone marrow mesenchymal stem cells surface antigen; cells after adipogenic osteogenesis inducing positive.Conclusions: After identification, the obtained cells are mesenchymal stem cells.Part II:The expression vector interference NMⅡ Constructed and cell screenedObjective:Prepared NMⅡ decreased expression of cell lines to provide cells for ALI treatment.Methods:By designing and constructing interference vector sequences between the previously identified transfected bone marrow mesenchymal stem cells, and then identified.Results:PLL recombinant vector after transfection to bone marrow mesenchymal stem cells obtained between NMⅡ decreased expression of bone marrow mesenchymal stem cellsConclusions:After identification, expressing NMⅡ protein reduced among the restructed vector transfected bone marrow mesenchymal stem cells.Part III:The efficacy and mechanism of the bone marrow stem cell in gene regulation NMⅡ treating inhalation lung injuryObjective: Detection NMⅡ gene regulation in bone marrow stem cells for treatment effect of inhalation injury model and mechanismMethods: First,established smoke inhalation lung injury model; experimental animal group divided into four group:control group, injury group, BMSC treated group, BMSC-Sh RNA group; rats were collected arterial blood to do gas analysis; using CM-Di I labeled bone marrow mesenchymal stem cell technology to detect cell distribution;rats bronchoalveolar lavage fluid collection; rat lung wet and dry ratio determination;HE detect lung inflammatory reactions 24hs;ELISA detect TNF-α and IL-10 factor in BALF; NMⅡ affect gene silencing on apoptosis of BMSC; NMⅡ affect gene silencing next BMSC cell secretion system.Results: Blood gas analysis found that rats reduced obviously damage hypoxemia, hypercapnia and acidosis after stem cell transplantation at 24 hs time point; the difference of treatment group and injury group was significant, inferred after BMSCs transplantation for ALI.Using CD-Di I labeled transfected NMⅡ-Sh RNA mesenchymal stem cells to inject inhalation injury model,1,4,7days, observing BMSCs cell homing. HE found damage interference group was significantly reduced.BALF El ISA detected in found the concentration of TNF-a reduced and IL-10 increased.Cell interference experiment group was significantly lower than that of the interference group caspase-3 expression. NMⅡ gene silencing effected BMSC paracrine system,HGF and VEGF increased than the normal group.Conclusions: Down-regulated gene expression NMⅡ stem cells could inhibit apoptosis and promote bone marrow mesenchymal stem cells homing to the site of injury, sit,reducing inflammation,it is more effective than traditional, simple to use mesenchymal stem cells.